Analytical validation of frataxin proteoforms in blood as biomarkers of Friedreich's ataxia

血液中 frataxin 蛋白质形式作为弗里德赖希共济失调生物标志物的分析验证

• 批准号: 10117295
• 负责人: Ian Alexander Blair
• 金额: $ 75.4万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10117295
• 关键词: Address; Affect; Age; Alleles; Alternative Splicing; Amino Acids; Biological; Biological Assay; Biological Markers; Blood; Blood Cells; Blood Platelets; Blood specimen; Cardiac; Cell Nucleus; Cessation of life; Clinical; Collection; Cultured Cells; Cytosol; Development; Disease; E protein; Erythrocytes; Friedreich Ataxia; Genes; Half-Life; High Pressure Liquid Chromatography; Immunoassay; Lateral; Length; Life; Mass Spectrum Analysis; Measurement; Methionine; Methods; Mitochondria; Mitochondrial Proteins; Monitor; Mutation; N-terminal; Pathology; Patients; Peptides; Plasma; Protein Isoforms; Proteins; Reaction; Resolution; Sampling; Sensitivity and Specificity; Serum; Site; Source; Specificity; Testing; Therapeutic Intervention; Therapeutic Trials; Time; Treatment Efficacy; Triplet Multiple Birth; Up-Regulation; Validation; Whole Blood; base; frataxin; healthy volunteer; liquid chromatography mass spectrometry; mitochondrial processing peptidase; nano; novel; novel therapeutic intervention; sample collection; stable isotope; therapy design; two-dimensional

ABSTRACT: ANALYTICAL VALIDATION OF FRATAXIN PROTEOFORMS IN BLOOD AS BIOMARKERS OF FRIEDREICH’S ATAXIA Friedreich’s ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50,000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood could not be used because frataxin was thought to be present in long-lived erythrocytes. An assay for analyzing frataxin in platelets, which have a half- life of 10 days, which would allow therapeutic interventions to be tested, was developed by the Blair and Lynch labs. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (nano- UPLC-MS/HRMS) and is monitoring three tryptic peptides from frataxin. The assay had 100 % sensitivity and specificity for discriminating between controls and FA cases but analyzing platelets on a routine basis is very challenging in many clinical settings. We have now discovered that in erythrocyte is in fact a novel proteoform of frataxin (isoform E) with 135-amino acids (76-210) and an N-terminally acetylated methionine residue. It arises through an alternative splice-site form that is used for the canonical full-length form of frataxin (1-210). There is three times as much isoform E in erythrocytes (26.7 ± 6.4 ng/mL) from the blood of healthy volunteers (n=10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). We recently found that both isoform E (8.5 ± 1.1 ng/mL) and mature mitochondrial frataxin (2.1 ± 1.1 ng/mL) are both reduced by > 70 % in blood from FA patients (n=29) when compared with healthy control subjects. Isoform E lacks a mitochondrial targeting sequence and so it is distributed to both cytosol and the nucleus when expressed in cultured cells. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood would make it possible to follow the progress of the disease and monitor the efficacy of therapeutic interventions. This will require the development of rigorously validated assays that address the pre-analytical and analytical issues that are relevant to the use of blood as a biological matrix for biomarker analysis rather than the more common use of serum or plasma. The present proposal addresses these issues under following specific aims: Aim 1: To conduct pre-analytical validation for collection and storage of mature frataxin and isoform E protein in whole blood. Aim 2: To conduct analytical validation of the method for quantifying mature mitochondrial frataxin and frataxin isoform E in whole blood. Aim 3: Validation of the mature frataxin and frataxin isoform E proteoform analysis in whole blood from controls, FA carriers, and FA cases from multiple sites.

摘要：血液中 frataxin 蛋白质组作为生物标志物的分析验证 弗里德里希共济失调 弗里德赖希共济失调 (FA) 是一种常染色体隐性遗传疾病，由内含子 GAA 三联体扩增引起 FXN 基因，导致线粒体蛋白 frataxin 表达减少。 FA 估计会影响 1 英寸 50,000 人的平均死亡年龄在 40 岁左右。目前尚无批准的 FA 治疗方法， 尽管涉及上调或替换 frataxin 蛋白的实验方法正在 已测试。 Frataxin 在血清或血浆中检测不到，并且不能使用全血，因为 Frataxin 据认为存在于长寿的红细胞中。一种分析血小板中 frataxin 的方法，其具有半 布莱尔和林奇开发了 10 天的生命周期，可以测试治疗干预措施 实验室。该测定基于稳定同位素稀释免疫纯化二维纳米超高 高效液相色谱/平行反应监测/高分辨率质谱（纳米 UPLC-MS/HRMS）并正在监测 frataxin 中的三种胰蛋白酶肽。该检测的灵敏度为 100% 区分对照和 FA 病例的特异性，但常规分析血小板非常困难 在许多临床环境中具有挑战性。我们现在发现红细胞中实际上是一种新的蛋白质形式 具有 135 个氨基酸 (76-210) 和 N 末端乙酰化蛋氨酸残基的 frataxin（异构体 E）。它 是通过另一种剪接位点形式产生的，该剪接位点用于 frataxin (1-210) 的规范全长形式。 健康志愿者血液中的红细胞 E 异构体含量 (26.7 ± 6.4 ng/mL) 是健康志愿者血液的三倍 (n=10) 与其他血细胞中存在的成熟线粒体 frataxin 相比 (7.1 ± 1.0 ng/mL)。 我们最近发现同工型 E (8.5 ± 1.1 ng/mL) 和成熟线粒体 frataxin (2.1 ± 1.1 ng/mL) 与健康对照受试者相比，FA 患者 (n=29) 血液中的两种物质均减少了 > 70%。 同工型 E 缺乏线粒体靶向序列，因此它分布到细胞质和细胞核中 当在培养细胞中表达时。特异性量化线粒体外和线粒体的能力 全血中 frataxin 的亚型将使追踪疾病的进展并监测 治疗干预的功效。这将需要开发经过严格验证的检测方法 解决与使用血液作为生物基质相关的预分析和分析问题 生物标志物分析而不是更常用的血清或血浆。本提案涉及 这些问题的具体目标如下： 目标 1：对成熟 frataxin 和同工型 E 蛋白的收集和储存进行分析前验证 在全血中。目标 2：对成熟线粒体的定量方法进行分析验证 全血中的 frataxin 和 frataxin 亚型 E。目标 3：验证成熟的 frataxin 和 frataxin 亚型 E 对来自多个地点的对照、FA 携带者和 FA 病例的全血进行蛋白质组分析。